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Expression patterns of adult liver markers. Detection of GSH depletion Conclusions Olaia Holgado, Maria Diez, Carles Callol-Massot, M. Luz Martinez-Chantar, Ainhoa Letamendia BIOBIDE S.L Donostia-San Sebastian, Spain PREDICTION OF HEPATOTOXICITY IN ZEBRAFISH Drug induced hepatotoxicity is one of the most important causes of drug withdrawal in the Drug Discovery process. The low correlation of the in vitro cytotoxicity studies, with human hepatotoxicity, especially in the early stages of Drug Discovery, make human hepatotoxicity difficult to be predicted. Therefore the finding of new methods and models to predict hepatotoxicity is a growing area of research. The zebrafish model has been extensively studied in the last decades for its ability to regenerate. Specifically, the liver is one of the organs with the highest regenerative capacity. Researchers over the past 10 years have characterized many of the mechanisms that occur during this process leading to an important knowledge of the signaling pathways that govern zebrafish liver function and development. Therefore, because of the functional as well as developmental similarities between the liver of zebrafish and mammals, we propose to analyze the potential of zebrafish for prediction of hepatotoxic agents. For this purpose, the following aspects have been studied: i) The stage at which adult liver markers are expressed has been identified. ii) Several techniques have been developed for assessing the hepatotoxicity of a compound in zebrafish. iii) These techniques will be validated by testing different drugs. iv) The results will be compared with those obtained in mammals. Our results indicate that zebrafish embryos could be used as an alternative model in the Drug Discovery process complementing the information of in vitro models and mammals and providing pharmaceutical industry of new assays which might decrease the current huge gap between in vitro and in vivo assays. Necrosis Glutathion (GSH) depletion Steatosis Q-PCR analysis Detection of steatosis In order to study the expression pattern of adult liver markers, we performed Q-PCR analysis of MAT1A, MAT2A, GNMT in zebrafish. Total RNA was isolated at 0, 5, 10, 15 and 25 dpf (days post-fertilization) using TRIZOL method. Approximately 20-100 embryos were pooled for each stage. cDNA was synthesized from total RNA using a high efficiency reverse transcriptase with oligo dT. Q-PCR analysis of MAT1A, MAT2A and GNMT was performed. Due to the differences in stages between the samples, no housekeeping gene was used for normalization. Then, three independent experiments were conducted to confirm the results. To evaluate the potential of zebrafish for the detection of liver necrosis, six compounds were selected (five positives and one negative) and tested at different concentrations for 24 or 48 hours. 10 embryos per compound and concentration were used. After treatment the embryos were anesthesized and the color intensity of the liver was quantified by using a specific software. Under conditions of oxidative stress, cells may transiently incorporate glutathione into proteins. Stressed cells incubated with BioGEE will also incorporate this biotinylated glutathione derivative into proteins, facilitating the identification of oxidation-sensitive proteins. Once embryos are fixed and permeabilized, glutathiolation levels are detected with a fluorescent streptavidin conjugate. Embryos were fixed with 4% paraformaldehyde, washed with PBS, incubated with a graded series of propylene glycol, and stained with Oil Red in 100% propylene glycol. Stained larvae were washed with decreasing concentrations of propylene glycol followed by several rinses with PBS and transferred to glycerol. |
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